CN1312858A - Bactericidal/permeability-increasing protein delation analogs - Google Patents

Abstract

Novel BPI deletion analogs are provided that consist of amino acid residues (10 through 193) of mature human BPI wherein the cysteine residue at BPI amino acid position (132) is replaced by another amino acid. Fusion proteins comprising these analogs are also provided, as are polynucleotides encoding these products, materials and methods for their recombinant production, compositions and medicaments of these products, and therapeutic uses for these products.

Description

Sterilization/permeability-increasing protein delation analogs

Background of invention

The invention provides the preparation and the pharmaceutical composition that contains these preparations of sterilization/permeability-increasing protein (BPI) delation analogs of new biologically active, new analogue wherein is characterised in that to have better stability and homogeneity and enhanced activity in vivo.

BPI is a kind of to resisting the vital Mammals hemocyte of invading micro-organism, is called isolating albumen in polymorphonuclear leukocyte (PMN or the bite the neutral cell) particle.Known BPI is in conjunction with lipopolysaccharides, and a kind of stimulation can cause the main ingredient of the powerful immunoreactive gram-negative bacteria outer membrane protein of septic shock (septic shock).By acid extraction coupled ion displacement chromatography [Elsbach, journal of biological chemistry, 254:11000 (1979)] or intestinal bacteria [E.coli] affinity chromatography [Weiss etc., blood, 69:652 (1987)], people BPI albumen obtains separating from PMNs.The BPI of Huo Deing is called natural B PI and has shown that it has powerful fungicidal activity to the wide spectrum gram-negative bacteria at this in this way.About 55,000 dalton of the molecular weight of people BPI (55kD).Complete people BPI protein sequence and this proteic DNA nucleotide sequence of coding are reported in Gray etc., journal of biological chemistry, and among Fig. 1 of 264:9505 (1989), it is for reference to mix the document at this.The aminoacid sequence of Gray etc. is shown among the SEQIDNO:1 here.U.S. Patent No. 5,198,541 (disclose at these only for reference) disclose coding BPI albumen, comprise the gene of recombinant BPI whole protein (being called rBPI) and BPI recombinant fragment and are used to express these proteic methods.

Proteolysis BPI N-end with about 25kD molecular weight has amphiphilic characteristic, contains the hydrophobic and hydrophilic area of alternative.This N-end of people BPI has the anti-microbial activity [Ooi etc., journal of biological chemistry, 262:14891-14894 (1987)] of natural 55kD people BPI whole protein.Opposite with the N-terminal portions, isolating people BPI PROTEIN C-stub area only demonstrates the Gram-negative biology slightly can detected anti-microbial activity [Ooi etc., The Journal of Experimental Medicine (J.Exp.Med), 174:649 (1991)].The terminal BPI fragment of the N-of a kind of about 23kD is called " rBPI ₂₃" prepared by recombination method and also keep anti-microbial activity to the Gram-negative biology [Gazzano-Santoro etc. infect.Immunity (Infect.Immun.) 60:4754-4761 (1992)].The terminal analogue of the N-of a kind of BPI, rBPI ₂₁, according to Horwitz etc. at the protein expression purifying, 8:28-40 has obtained preparation described in (1996).

It is high degree of specificity that the bactericidal effect of BPI is reported to the gram-negative bacteria kind, for example, at Elsbach and Weiss, inflammation: ultimate principle and clinical correlation, volumes such as Gallin, the 30th chapter, Raven publishes described in the company limited (1992).The target cell specificity of this report is thought BPI to lipopolysaccharides (LPS), the result of the strong absorption of the biological adventitia of Gram-negative (or bag quilt) specific component.Though BPI it has been generally acknowledged that other microorganism, comprise that yeast and more high eukaryotic cell are nontoxic, but recent findings, as discussed below, the BPI protein product also shows activity to Gram positive cell, mycoplasma, mycobacterium, fungi, protozoon and chlamydozoan.

Though BPI kill gram-negative bacteria really cutter system do not illustrate fully as yet, think BPI must be at first BPI albumen by positively charged and LPS go up on the surface that static between the electronegative site and hydrophobic interaction be attached to this bacterium.Because the powerful inflammatory reaction of its stimulation promptly discharges (inflammation) medium by host's inflammatory cell, this can finally cause irreversible interior toxicogenic shock, so LPS is called " intracellular toxin " again.BPI be reported to the part that tool is toxic and biological activity is the strongest of LPS, lipid A combination.

In the gram-negative bacteria of susceptible, BPI has interrupted the LPS structure in conjunction with thinking, the activation of the bacterial enzyme of cause degrading phosphatide and peptidoglycan, thus change the saturating property of epicyte, and start the incident that finally causes necrocytosis.[Elsbach and Weiss (1992) see above].BPI thinks and works two periods.First is called inferior causing death period, and it is characterized by grows immediately stops, and penetrates the bacterium enzyme of adventitia and selectivity activation hydrolytic phosphatide and peptidoglycan.The bacterium in this period can be incubated in the substratum that replenishes serum protein and obtain saving [Mannion etc., Journal of Clinical Investigation (J.Clin.Invest.) 85:853-860 (1990)].For a long time with bacterial exposure after BPI, be defined as and can not take place growth inhibiting second period by serum protein reverses, it is characterized in that physiology and structural modification widely, comprise obvious damage to inner membrance.

BPI and LPS initial combines and causes the variation organized, and this may be to come from by in conjunction with Mg ⁺⁺And Ca ⁺⁺And in conjunction with the anionic group of LPS, adventitia is stablized in this combination usually.BPI is attached to the gram-negative bacteria adventitia and causes hydrophobic drug such as dactinomycin to penetrate adventitia rapidly.The combination of BPI and subsequently gram-negative bacteria kill the length that partly depends on the LPS polysaccharide chain at least, " smooth type " biological " rough type " the biological bactericidal effect to BPI that has short O-chain that wherein has longer O-chain has stronger resistance [Weiss etc., Journal of Clinical Investigation, 65:619-628 (1980)].In first period of this BPI effect, penetrating at BPI of gram-negative bacteria exoperidium is reversible [Weiss etc., Journal of Immunology, 132:3109-3115 (1984)] when dissociating (a kind of high density divalent cation and new LPS synthetic process of needing).Yet, by recovering the integrity of bag quilt, the forfeiture of the survival of Gram-negative bacteria does not obtain reversing, show what this germicidal action was mediated by other damage of inductive in the target biology, and this damage may be positioned at (Mannion etc. on the plasma membrane, clinical, research magazine, 86:631-641 (1990)).Particular studies to this possibility has been presented on the mole foundation, BPI has identical plasma membrane carrier inhibit feature (In ' tVeld etc. with PXB at least, infect and immunity, 56:1203-1208 (1988)), but the dependency between definite mechanism and this carrier and complete biological study also illustrated.

Shown that the BPI protein product (comprises BPI albumen natural and the reorganization preparation; Natural, synthetic and recombinate BPI protein polypeptide fragment with biologic activity; Have BPI albumen or its segmental polypeptide variants of biologic activity, comprise hybrid fusion protein and dimer; Have the BPI albumen of biologic activity or the analogue of its fragment or varient, comprise halfcystine alternate analogue; With the BPI derived peptide) have a multiple favourable activity.Known BPI protein product has bactericidal effect to gram-negative bacteria, as at United States Patent(USP) Nos. 5,198, and 541 and 5,523, described in 288, mix at this that the two is for reference.Simultaneously known BPI protein product strengthens antibiotic effect in gram-negative bacteria infects, as in U.S. Patent No. 5,523,288 and corresponding international open No.WO 95/08344 (PCT/US 94/11225) described in, mix for reference at this.Known BPI protein product has bactericidal effect to gram-positive bacteria and mycoplasma simultaneously, and strengthen the effect of microbiotic in gram-positive bacteria infects, as in U.S. Patent No. 5,578,572 with described in the corresponding international open No.WO 95/19180 (PCT/US95/00656), quotes for reference at this.Also further known BPI protein product demonstrates anti-mycotic activity and strengthens the activity of other anti-mycotic agent, as in U.S. Patent No. 5,627,153 and corresponding international openly described in the No.WO 95/19179 (PCT/US 95/00498), and owning together, the common unsettled U. S. application series No.08/621 that is filed on March 21st, 1996,259 (it is again the U. S. application series No.08/504 that is filed on July 20th, 1994 conversely, 841 part continues) and corresponding international disclose to further describe among Nos.WO 96/08509 (PCT/US 95/09262) and the WO 97/04008 (PCT/US 96/03845) be anti-fungus peptide, it is for reference to introduce all documents.Also further known BPI protein product demonstrates the protozoacide activity, as in U.S. Patent No. 5,646,144 and corresponding international open No.WO 96/01647 (PCT/US 95/08624) described in, it is for reference to be incorporated herein document.Known BPI protein product demonstrates anti--chlamydozoan activity, as at the unsettled U. S. application series No.08/694 that is filed on August 9th, 1996 that own together, common, 843 with described in the corresponding international open No.WO 98/06415 (PCT/US 97/13810), and all documents that are incorporated herein are for reference.At last, known BPI protein product demonstrates the mycobacteria activity, as own together, the common unsettled U. S. application series No.08/626 that is filed on April 1st, 1996,646 (it is again the U. S. application series No.08/285 that is filed on August 14th, 1994 conversely, 803 part continues, the latter becomes the U. S. application series No.08/031 that submitted on March 12nd, 1993 again, 145 part continues) and corresponding international disclosing described in the No.WO 94/20129 (PCT/US 94/02463), the document of all introducings is for reference.

The BPI protein product in the mankind to the circulation in endotoxic effect, comprise TNF, IL-6 and endotoxic effect are described in United States Patent(USP) Nos. 5,643,875 and 5,753,620 and corresponding international open No.WO 95/19784 (PCT/US 95/01151) in, it is for reference to introduce all documents.

Known BPI albumen is useful for treating in the following specificity state of an illness simultaneously, for example people meningococcus mass formed by blood stasis (meningococcemia) is (as owning together, the common unsettled U. S. application series No.08/644 that is filed on May 10th, 1996,287 and corresponding international openly described in the No.WO 97/42966 (PCT/US 97/08016), be incorporated herein for reference), people's traumatic hemorrhage is (as owning together, common unsettled U. S. application series No.08/862,785, be filed in the U.S. series No.08/652 on May 23rd, 1996,292 part continuity, present U.S. Patent No. 5,756,464 and corresponding international openly described in the No.WO 97/44056 (PCT/US 97/08941), it is for reference to quote all documents at this), burn is (as in U.S. Patent No. 5,494,896 and corresponding international openly described in the No.WO 96/30037 (PCT/US 96/02349), it is for reference to be incorporated herein these two pieces of documents), the perfusion of local asphyxia/again (reperfusion) damage is (as in U.S. Patent No. 5,578, described in 568, be incorporated herein for reference) and hepatotomy (as owning together, the total unsettled U. S. application sequence No.08/582 that is filed on March 16th, 1998, described in 230, this application is the continuity application to the same train that is filed on January 3rd, 1996 number, it is again the U. S. application series No.08/318 that is filed on October 5th, 1994 conversely, 357 continuity, the latter is again the U. S. application series No.08/132 that is filed on October 5th, 1993,510 part continuity, and corresponding international open No.WO 95/10297 (PCT/US 94/11404), it is for reference that all introduce document).

Simultaneously in the known BPI protein product and the antithrombotics activity of exogenous heparin, as in U.S. Patent No. 5,348, described in 942, be incorporated herein for reference, and to the treatment chronic inflammatory diseases be useful as similar rheumatism and reactive arthritis, as in U.S. Patent No. 5, described in 639,727, quote for reference at this, and the disease that is used to suppress vasculogenesis and treat associated angiogenesis comprises malignant tumor, eyepiece retinopathy (ocular retinopathy) and endometriosis (endometriosis) are as owning together, common unsettled U. S. application series Nos.08/435,855,08/466,624 and 08/466,826 with described in the corresponding international open No.WO94/20128 (PCT/US 94/02401), and it is for reference at this that all introduce documents.

Simultaneously known BPI albumen is used for antithrombotic method, as in U.S. Patent No. 5,741,799 and corresponding international open No.WO 97/42967 (PCT/US 97/08017) described in, be incorporated herein for reference.

United States Patent(USP) Nos. 5,420,019 and 5,674,834 and corresponding international open No.WO94/18323 (PCT/US 94/01235) (it is for reference that all introduce documents) disclose halfcystine and make the BPI polypeptide that obtains that the formation of dimerization and halfcystine addition compound is had resistance with other amino acid replacement in amino acid position 132 or 135.It discloses simultaneously at BPI amino acid position 193 and has stopped the expression product that the terminal BPI fragments of N-cause having the C-terminal unhomogeneity of minimizing.

Interesting is Capodici and Weiss be in immunology, the report among the 156:4789-4796 (1996), i.e. encoding mature BPI amino-acid residue 1 to 193 (BPI _1-193) and residue 13 to 193 (BPI _13-193) in-vitro transcription/translation product of DNA show the combination of the similar LPS-dependency of fixed L PS.

Still exist such necessity in the art, promptly (preparation) have the BPI protein product preparation of higher biologic activity, particularly those have the preparation of biologic activity in enhanced stability, homogeneity and/or the body.

Summary of the invention

The invention provides new BPI delation analogs and goods thereof with biologic activity, it is characterized in that enhanced stability and homogeneity, for example comprise N-terminal and C-terminal unhomogeneity and enhanced biologic activity that resistance that dimerization and halfcystine adducts are formed and recombinant products reduce, these characteristics make it be applicable to that highly treatment and diagnosis use.New BPI delation analogs is for being encoded into the expression product of acquaintance BPI (SEQ ID NO:2) amino-acid residue 10 to 193DNA, and the halfcystine of position 132 is wherein replaced by different amino acid, is preferably nonpolar amino acid such as Serine or L-Ala.In embodiment preferred, called after " rBPI (10-193) C132A " or " rBPI (10-193) ala ¹³²" in, 132 halfcystine is substituted by L-Ala.

The new purifying and the isolating polynucleotide sequence (for example, DNA or RNA) of these BPI protein products of encoding have been the present invention further provides; The materials and methods that is used for its reorganization preparation comprises carrier and comprises the host cell of this DNA; The more stable pharmaceutical composition that comprises these BPI protein products; The improved methods of treatment of using these compositions or while and other therapeutical agent to use separately.The BPI delation analogs of the present invention that also has that relates to simultaneously is used for the treatment of the use in the medicament of benefit patient from use the BPI protein product in preparation.

Others that the present invention is numerous and advantage will be conspicuous to those of skill in the art when the present invention describes in detail below considering, this detailed description has been described present its embodiment preferred.

The accompanying drawing summary

Fig. 1 has described and has used rBPI (10-193) C132A or rBPI ₂₁The elevation of blood pressure that is determined as area under the curve (area under curve (AUC)) that the back takes place.

Detailed Description Of The Invention

The invention provides by the new BPI delation analogs that becomes acquaintance BPI amino acid residue 10 to 193 (listing in SEQ ID NO:2) to form, the cysteine residues of BPI amino acid position 132 wherein is preferably nonpolar propylhomoserin such as serine or alanine and is substituted by other amino acid. 132 position cysteines wherein are by preferred embodiment called after rBPI (10-193) C132A or rBPI (10-193) ala1a that alanine substituted¹³²。

BPI protein product rBPI₂₁For being encoded into the expression product of acquaintance BPI amino acid residue 1 to 193 DNA, 132 residue cysteines are wherein substituted by alanine, as in U.S. Patent No. 5,420, described in 019. Be used for by reaching more high-cell density and Geng Gao rBPI₂₁The titre recombination method prepares rBPI₂₁Fermentation process in variation also caused the obvious increase of the terminal inhomogeneity of purified product propylhomoserin. In some fermentation circulation, the purified product of observing up to about 20% is the kind with BPI amino acid/11 0-193, rather than 1-193 amino acid of coding. The SDS-PAGE gel of 500 liters of fermented samples in the fermentation cyclic process shows that this 10-193 kind appears at last 2-3 days of this circulation, and wherein maximum comes across and gathers in the crops the same day. Further studies show that rBPI₂₁Produce digestion product with CHO-K1 cell homogenates incubation, point out the proteinase activity relevant with this cell to participate in (this process). For the mode simulated albumin enzymatic activity with control, rBPI₂₁With Aminopeptidase M and elastoser incubation. RBPI₂₁Digestion has resistance to Aminopeptidase M, but elastoser is rapidly with rBPI₂₁Convert 40% BPI (8-193) and 60% BPI (10-193) to.

As described herein, the rBPI of stable uniform (10-193) C132A preparation obtains preparation by proteolysis and recombination method. The BA of purifying and this albumen of test. Measure at several extracorporeal biologies, test in 2 different animal efficiency Model and pharmacokinetics and the toxicologic study with relatively rBPI (10-193) C132A and rBPI₂₁ As described in the embodiment 5-7, rBPI (10-193) C132A and rBPI₂₁Below relatively in have similar external activity, comprise with radial diffusion and the little dilution of culture medium (microdilution) sterilization of Escherichia coli J5 and measuring, radial diffusion with L-shape staphylococcus aureus (Staphylococcus aureus) is measured, with the competition of Escherichia coli J5 LPS in conjunction with mensuration with among the LPS of RAW and THP1 cell and measure. As if other experiment that is described among the embodiment 5 shows that rBPI (10-193) C132A approximately is rBPI at the LPS with speed turbidimetry (nephelometry) in measuring₂₁The twice of intensity. As described in example 8 above, the rBPI of purifying (10-193) C132A and rBPI₂₁In dosage reaches 120mg/kg/ days three days rat GLP toxicologic study, have similar toxicity profile and be to have similar pharmacokinetics in the 2mg/kg rat at dosage. The experiment of describing among the embodiment 8 is also shown in the mouse endotoxin inoculation model, and rBPI (10-193) C132A seems at least than rBPI in two researchs₂₁Active strong twice, and in mouse causes death bacterium blood model, rBPI (10-193) C132A and rBPI₂₁Effectively same. Except experiment in the body of rat consciously, perfusion rBPI ₂₁40 compare with vehicle Control with the dosage of 50mg/kg and to cause that blood pressure is significant temporary transient to descend, and the rBPI of same dose (10-193) C132A does not cause the statistically evident temporary transient decline of blood pressure compared with the control. Therefore, with perfusion rBPI₂₁As if compare, perfusion rBPI (10-193) C132A provide the decline of side effect in the blood.

The invention further relates to the fusion of at least a portion of rBPI (10-193) C132A and at least a other polypeptide.The case description of this hybrid fusion protein is in U.S. Patent No. 5,643,570 and corresponding international open No.WO 93/23434 (PCT/US 93/04754) in (it is for reference to be incorporated herein all documents) and comprise hybrid fusion protein, its N-terminal comprises that BPI albumen or its bioactive fragment and its C-terminal comprise a constant region or its allelic variant of heavy chain immunoglobulin at least.

The invention still further relates to the new BPI delation analogs of code book invention or the purifying and the isolating polynucleotide sequence (for example, DNA or RNA) of fusion rotein; The expression vector that contains these polynucleotide, polynucleotide wherein preferably be operably connected to endogenous or the heterogenous expression regulating and controlling sequence on; Protokaryon or eukaryotic host cell with DNA of the present invention or carrier stable transfection or conversion; With the method that is used for the new delation analogs BPI protein product of the present invention reorganization preparation, for example, wherein host cell is incubated in the substratum of proper nutrition and the method for from this cell or substratum, separating delation analogs BPI protein product.These polynucleotide sequences or carrier can be chosen coding 27-amino acid BPI leader sequence and mouse light chain polyadenylation signal wantonly.

The present invention recombinates the new BPI delation analogs of preparation can be according to being described in U.S. Patent No. 5,439,807 and corresponding international open No.WO 93/23540 (PCT/US 93/04752) (being incorporated herein for reference) in method prepared.U.S. Patent No. 5,439,807 disclose the method that in culture purifying is expressed the recombinant BPI protein product of justacrine in the genetic transfection mammalian host cell, and disclose people and how to prepare and be applicable to the recombinant BPI product that mixes in stable, the homogeneous pharmaceutical preparation in a large number.

The present invention further provides the more stable pharmaceutical composition that comprises new BPI delation analogs and separately with these compositions or the improved methods of treatment used simultaneously with other therapeutical agent.Expect that this composition can be used for known BPI protein product, in any therepic use of those products that comprise above being discussed.

Usually, use the BPI protein product, comprise that the BPI delation analogs is preferably finished with pharmaceutical composition, said composition comprises BPI protein product and pharmaceutically acceptable thinner, adjuvant or carrier.The BPI protein product can use separately or with known tensio-active agent, other chemotherapeutics or other known anti--chlamydial medication combined using.Contain BPI protein product (for example, rBPI ₂₃) stable pharmaceutical composition comprise citric acid buffering salt (5 or the 20mM Citrate trianion, 150mM NaCl, pH5.0) the BPI protein product of middle 1mg/ml concentration is comprising poloxamer 188 (the Pluronic F-68 of 0.1% weight, BASF, Parsippany, NJ) and the polysorbate (polysorbate) 80 of 0.002% weight (Tween 80, ICIAmericas company limited, Wilmington, DE or JT Baker, Phillipsburg, NJ).Another kind contains BPI protein product (rBPI for example ₂₁) stabilizing pharmaceutical composition be included in the BPI protein product of 2mg/ml concentration in 5mM Citrate trianion, 150mM NaCl, 0.2%poloxamer 188 and 0.002% polysorbate80.This preferred combination is described in United States Patent(USP) Nos. 5,488,034 and 5,696,090 and corresponding international open No.WO 94/17817 (PCT/US 94/01239) in, it is for reference to be incorporated herein all documents.As the U. S. application sequence No.08/586 that submits on January 12nd, 1996,133 (it is again the U. S. application series No.08/530 that submits to September 19 nineteen ninety-five, 599 part continuity, the latter is again the U. S. application series No.08/372 that submits to January 13 nineteen ninety-five, 104 part continues) and corresponding international disclosing described in the No.WO 96/21436 (PCT/US 96/01095) (it is for reference to be incorporated herein all documents), other poloxamer preparation with enhanced activity BPI protein product also can use.

But comprise the therapeutic composition system or the topical application of BPI protein product.The systemic application approach comprises that oral cavity and enteron aisle use outward, (with powdered drug or atomizing or atomization drug solution) or applied dermally after comprising intravenously, intramuscular or subcutaneous injection (comprise in the store chamber (depot) and discharging), intraocular and eyeball, in the sheath, in the intraperitoneal (for example, intraperitoneal lavation), lung for long-term.The U. S. application series No.08/962 that improved atomization preparation is described in and owns together, submit to 31 days common unsettled October in 1997,217 and corresponding international open No.WO98/19694 (PCT/US 97/19850) in, it is for reference to be incorporated herein two documents.

When the enteron aisle external administration, BPI protein product composition generally with the injection of following dosage, comprises μ g/kg to 100mg/kg every day 1, preferred every day 0.1mg/kg to 20mg/kg, more preferably 1 to 20mg/kg/ day and most preferably be 2 and arrive 10mg/kg/ days.This treatment can be passed through with same dose or minimizing or increase dosage continous pouring every day or intermittent injection or perfusion, and for example 1 to 3 day, and determine this dosage by treating the doctor in addition.When intravenously was used, the BPI protein product preferably continued to pour into and used by the simple perfusion back of beginning.Preferred intravenous therapy scheme is to continue the intravenously perfusion with 20mg/kg/ days dosage behind 1 to the 20mg/kg simple intravenously perfusion BPI protein product, continues to reach a week.Particularly preferred intravenous dosages scheme is that 1 to 4mg/kg initial simple intravenously perfusion back continues the intravenously perfusion with 1 dosage that arrives 4mg/kg/ days, continues to reach 72 hours.

Local approach comprises with following form to be used, be ointment, emulsifiable paste (cream), gel, eye dropping liquid or ointment (as own together, the common unsettled U. S. application series No.08/557 that is filed in November 14 nineteen ninety-five, 289 and U.S. Patent No. 5,686,414 reach described in corresponding international open Nos.WO 97/17990 (PCT/US 96/18632) and the WO 97/17989 (PCT/US 96/18416), it is for reference that all introduce documents), ear dropping liquid, suppository, washing lotion (for example, bathing a wound) or medicated shampoo.For example, for the topical application of drop form,, can use every day 1 time or repeatedly about 10 to 200 μ L BPI protein product compositions according to treatment doctor's judgement.

Those skilled in the art can easily optimize effective dose and the application program that comprises BPI protein product therapeutic composition according to the clinical state of an illness of good medical practice and single patient.

Will be seen that by following illustrative embodiment others of the present invention and advantage.Embodiment 1 describes the structure of coding rBPI (10-193) C132A expression vector pING 1742.Embodiment 2 describes the screening with the conversion of 1742 pairs of Chinese hamster ovary celIs of pING and secretion rBPI (10-193) C132A production peak clone.Embodiment 3 is described in preparation and the purifying of rBPI (10-193) C132A in 2-L and the 500-L fermentor tank.Embodiment 4 has described rBPI (10-193) C132A and rBPI ₂₁The biochemical character analysis.Embodiment 5,6 and 7 describes respectively and rBPI ₂₁Compare below in the determination and analysis external LPS-in conjunction with activity, comprise competition in conjunction with determination and analysis and with among the LPS of speed tuurbidimetry, fungicidal activity and rBPI (10-193) C132A and the determination of activity mixture form the analysis of speed.The embodiment Final 8 has transferred the activity in vivo of rBPI (10-193) C132A.

Embodiment 1

The structure of expression vector pING1742

RBPI (10-193) C132A expression vector, pING 1742, made up by following.At first by connecting following fragment construction of expression vector pING 4115, comprise BamH I-Bsa I fragment that contains pING 3174neo gene and the Bsa I-Xho I fragment that contains the CMV promotor and from the rBPI of pING 4144 ₂₁Gene and contain Xho I-BamH I fragment from pING 4537 mouse (kappa) light chain 3 ' non-translational region (pING 3174, and pING 4144 and pING4537 are described in U.S. Patent No. 5,420, in 019, are incorporated herein for reference).The pING4155 carrier that obtains contains the coding rBPI that is fused on human IgG enhanser, people CMV promotor and mouse (kappa) light chain 3 ' non-translational region ₂₁Gene.Its neo gene that contains the neomycin phosphotransferase of encoding simultaneously is used for screening to microbiotic Geneticin (Geneticin ^) (G418) transformant of resistance.

Hind III-Hind III the fragment that contains the pING4155 0.7kbp of people Ig enhanser by deletion prepares carrier pING 1732.Then, from pING 1732, delete 27 Nucleotide of encoding mature rBPI21 partial amino-acid 1 to 9 by stack PCR mutagenesis with following primer: primer 1:5 '-CTGCTCTAAAAGCTGCTGCAG-3 ' (SEQ ID NO:3) primer 2: 5 '-CCAGGCCCTTCTGGGAGGCCGCTGTCACGGCGG-3 '

(SEQ ID NO:4) primer 3:5 '-GCCGTGACAGCGGCCTCCCAGAAGGGCCTGGAC-3 '

(SEQ ID NO:5) primer 4:5 '-CTGGGAACTGGGAAGCTG-3 ' (SEQ ID NO:6) synergetic complementary primer 2 and 3 mixes the disappearance of the 27bp Nucleotide of coded amino acid 1 to 9, and primer 1 and 4 is encoded respectively and is positioned at the Nucleotide of pING 1732 distinctive Sa II and EcoR I site upstream and downstream.At first, the combination with Oligonucleolide primers 1 and 3 and 2 and 4 obtains these fragments by pcr amplification.After obtaining these independent fragments, itself and primer 1 and 4 are annealed, extended and increase once more.Thereby the fragment of this amplification is then with the digestion of Sal I and EcoR I and be cloned among the pING 1732 of Sal I-EcoR I digestion and obtain plasmid pING 1742.

In order to confirm that Sal I-EcoR I zone of order-checking pING 1742 takes place not have sudden change in the PCR process.Do not observe variation in sophisticated BPI coding region.Yet, discovery two base pairs change in the DNA of coded signal sequence (ACC-＞GCT), thus cause being converted to Ala by Thr with respect to the amino acid of maturation protein-6 position.

Embodiment 2

Conversion with 1742 pairs of Chinese hamster ovary celIs of pING

By growing in following Ex-Cell 301 substratum that CHO-K1 cell (American type culture collection (ATCC) license number No.CCL 61) are adapted to serum-free.To be incubated at CHO-K1 cell in the Ham ' s F12 substratum with tryptic digestion, centrifugal and be resuspended in Ex-Cell 301 substratum.Cell cultures was gone down to posterity once by three days in the 125ml of 100rpm culturing bottle and with per two days of 125ml or 250ml culturing bottle.

The Chinese hamster ovary celI that these Ex-Cell 301-are adapted to by electroporation with in addition transfection of pING 1742.Before the transfection, with the Not I digestion pING 1742 of this plasmid of linearizing.Reclaim back 48 hours with cell with about 10 ⁴Individual cells/well is planted in containing and is replenished 0.6mg/mL G418 (Life Technologies, Gaithersburg is MD) in the 96 hole flat boards of Ex-Cell 301 substratum.In about 2 whens week, contain the existence of BPI-proteins C reactive in about 250 hole supernatant liquors of single colony by the ELISA screening with anti--BPI monoclonal antibody.

15 clones that will have high expression level are transferred in the 24 hole flat boards that contain Ex-Cell 301 substratum.In order to screen productive rate, with cell cultures in contain replenish with 24 hole flat boards of 2%FBS and the aseptic S-Sepharose globule of 40 μ L Ex-Cell substratum in 10 days, take out globule then, with low salt buffer (0.1M NaCl in the 10mM sodium acetate, pH 4.0) flushing, and in identical damping fluid, use 1.5M NaCl wash-out BPI.Measure the level of secretion rBPI (10-193) C132A with ELISA.The Western engram analysis of the eluate in 12% non--reductibility sds gel demonstrates migration than rBPI ₂₁Slightly fast obvious band.

The first eight colony that output is the highest is transferred in the aseptic 125mL triangular flask (Erlenmeyer) and is incubated in the Ex-Cell substratum.Replenish with in the triangular flask of the aseptic S-Sepharose globule of 2%FBS and 1% (v/v) Ex-Cell 301 substratum and assess its productive rate once more by it being incubated at contain.Wash-out rBPI (10-193) C132A and measure the level of rBPI (10-193) C132A with HPLC in the S-Sepharose globule from mix substratum.Picking out the clone 139 who is arranged in production peak is used for further cultivating and the product preparation.

Embodiment 3

Preparation and the purifying of rBPI (10-193) C132A

By 2 or research with fermentor tank (Biolafitte, St.Germain en Laye, France) and then (ABEC, Allentown cultivate clone's 139 cells in PA) and prepare a large amount of rBPI (10-193) C132A and be used for signature analysis at 500 liters of ABEC fermentor tanks.Protein product available from 2 liters of fermentor tanks is used for following in vitro study, and is used for animal toxicology and Study on Efficiency available from the product of 500 liters of fermentor tanks.A. the cultivation in 2 liters of fermentor tanks

Thereby clone's 139 cells are gone down to posterity in the rotating and culturing bottle that adds large volume with 1%FBS Ex-Cell substratum gradually up to reaching enough volume and cell concn with about 2 * 10 in containing to replenish ⁵Individual cell/mL inoculates 2 liters bio-reactor.In 37 ℃, 150rpm is incubated in three 2 liters the fermentor tank with cell, culture medium supplemented wherein 1% FBS, pH is 7.2, dissolved oxygen is maintained at 5-10%.With 1.5% (V/V) add large-scale aseptic SP-Sepharose globule (Pharmacia and Upjohn, Piscataway, NJ).Initial glucose level is approximately 3.5g/L and mend the glucose pulsed to 3g/L every day during the fermentation.Stopped fermentation in the time of 238 hours, this moment, the viability of cell was 63%, 80% and 84%.

After the fermentation, gather in the crops the globule of every fermentor tank, make its deposition (settle) thus and remove the cellular component and the faint bonded impurity of globule for several times with the 10mM sodium phosphate of pH7.0/0.15M NaCl flushing.With the globule dress post of flushing, with the 10mM sodium phosphate of pH7.0, the 0.25mMNaCl flushing, and with containing 0.8M NaCl, the same buffer wash-out of 5mM glycine.Eluate is used aseptic injection water (WFI) wash-out of 3 times of volumes then, CM-spherodex post (Sepracor packs into, Marlborough, MA) also use the 10mM sodium phosphate of pH7.0,0.25MNaCl to wash, sodium acetate, 0.2M NaCl with pH4.0 20mM washes then, then with sodium acetate, the 0.3M NaCl flushing of pH4.0 20mM, and with sample wash-out in addition in identical damping fluid.With 10, the Centricon film that 000MW blocks (Amicon, Beverly, MA) concentrate after, will pack into in pH5.0 5mM Trisodium Citrate, the 0.15M NaCl equilibrated Sephacryl S-100 post (Pharmacia and Upjohn) from the eluate of CM post.The part that will contain rBPI (10-193) C132A that identifies by 280nm place absorption value merges, and be concentrated into 1.9mg/mL also with 0.002% polysorbate fat 80 (JT Baker with the Amicon filter membrane, Phillipsburg, NJ), 0.2%poloxamer 188 (PluronicF-68, BSAF, Parsippany NJ) is prepared.Final preparation is with 0.2 μ m membrane filtration degerming.B. the cultivation in 500 liters of fermentor tanks

Thereby clone's 139 cells are gone down to posterity in the no Pp63 glycophosphoproteins Ex-Cell substratum that contains 1%FBS a series of increase the revolving bottle of volume gradually be provided for 35 liters of Bellco revolving bottle (Bellco Glass, Vineland, NJ) inoculum, the latter provides inoculum for 500L ABEC fermentor tank again conversely.With cell cultures in no Pp63 glycophosphoproteins but replenish in the complete Ex-Cell substratum with 1%FBS, extra glucose (to 10g/L) and glutamine (to 10mM).Fermentor tank moves in feed supplement-batch-wise mode, and therein in service is replenished pulse and a glucose/glutamine pulse is carried out with a 0.5%Primatone RL.500 liters of fermentor tanks are inoculated back 24 hours and are added 5 to 6 liters of large-scale SP-Sepharose globules.Sodium bicarbonate manual adjustment pH to 7.0 with 10%, oxygen be controlled at 5% and temperature be controlled at 37 ℃.Keep the stirring of 25rpm with the pulpous state thruster of two three-swords.Stop when fermentation runs on 184 hours, this moment, the viability of cell was 90%.

As 2 liters of above-mentioned fermentations, fermentation relief globule deposits and uses then the flushing of less salt (0.1M) phosphoric acid buffer for several times.Except the virally inactivated step that comprises pH3.0 into behind S-Sepharose globule wash-out and second CM-spherodex post were comprised into as enrichment step, purification step and the above-mentioned condition that is used for 2 liters of samples were similar.For second CM post, with the long-pending WFI dilution eluate of triploid, regulate pH to 5.0, with 20mM sodium acetate, the 0.3M NaCl balance of pH5.0 and wash this post and in identical damping fluid with the 1.0MNaCl elution samples.With the 5mM Trisodium Citrate of pH5.0,0.15M NaCl wash-out rBPI (10-193) C132A from the SephacrylS-100 post, be adjusted to 2mg/mL, and through 0.2 μ m membrane filtration.Use 0.002% polysorbate80 then, 0.2%poloxamer 188 preparation rBPI (10-193) C132A, sterile filtration, and in the 10mL I type vial of packing into.

Embodiment 4

The biochemical character of rBPI (10-193) C132A is analyzed the albumen of A. from 2 liters of fermentations

RBPI (10-193) the C132A product of observing purifying among the embodiment 3 is that migration is than rBPI at sds polyacrylamide gel electrophoresis (SDS-PAGE) ₂₁The single band that band is fast slightly, this and rBPI ₂₁Terminal 9 aminoacid deletion of N-are consistent.Sequential analysis shows that rBPI (10-193) C132A contains the N-end sequence of expectation: SQKGLDYASQQGTAALQKEL.Observe two kinds of components during mass spectroscopy (ESI-MS), one is 20,470 daltonian molecular weight, this and 20 of rBPI (10-193) C132A that estimates, 472 Dalton molecular weights are consistent, and second has 20,225 daltonian molecular weight, these 20,258 daltonian molecular weight with the rBPI (10-191) that estimates are consistent.RBPI (10-193) C132A and rBPI ₂₁Ion-exchange HPLC figure (HeWlett-Packard, Model 1050, Palo Alto CA) all shows single peak and having the identical residence time.B. from the albumen of 500 liters of fermentations

When carrying out SDS-PAGE, rBPI (10-193) C132A is for moving than rBPI ₂₁The single band that band is fast slightly.When carrying out mass spectrum (analysis), the main band of one 20,471 Dalton molecular weight is arranged, 20,474 Dalton molecular weights of the expectation of itself and rBPI (10-193) C132A are consistent, and two accessory constituents, a molecular weight is 20,668 dalton, N-acetyl hexylamine (estimated molecular weight 20,677 dalton) is consistent with adding, and another molecular weight is 20,843 dalton add that with interpolation N-acetyl amine hexose (estimated molecular weight is 20,839 dalton) is consistent.At preparation rBPI ₂₁In the process, often observe one and have the similar components of adding N-acetyl hexylamine.

When carrying out reversed-phase HPLC (Shimadzu, capital of a country, Japan), rBPI (10-193) C132A and rBPI ₂₁All wash-out becomes a main peak and an accessory peak.Yet, the corresponding rBPI during rBPI (10-193) C132A peak contrasts ₂₁Peak wash-out a little earlier comes out.The glycosylation form that the most possible representative of secondary peaks in rBPI (10-193) the C132A figure identifies in mass spectrum.RBPI (10-193) C132A and rBPI ₂₁Ion-exchange HPLC figure all show single peak and have the similar residence time.

Carried out the trypsinase mapping analysis according to traditional method.The rBPI of acetone precipitation ₂₁Or rBPI (10-193) C132A at first uses dithiothreitol (DTT) (DTT) processing back to handle and use then trypsin treatment with iodine ethamine.The product of trypsin treatment is analyzed by HPLC (Beckman Model 126) with C18 post (BeckmanUltrasphere).At rBPI ₂₁In, there are 2 to be derived from terminal trypsinase fragment (T1 and Ala-T1) to the N-of the inaccurate cutting of leader sequence.As estimating, except disappearance N-terminal fragment, trypsin hydrolyzing figure and the rBPI of rBPI (10-193) C132A ₂₁Similar.

Embodiment 5

The external LPS-of rBPI (10-193) C132A is in conjunction with active A. in the competition binding analysis (activity)

Assessment is according to purifying rBPI (10-193) C132A and the rBPI of embodiment 3A preparation in the competition binding analysis ₂₁RBPI with mark ₂₁Competition is in conjunction with the ability of LPS.In brief, with fixed concentration (0.5nM) ¹²⁵The rBPI of I-mark ₂₁With unlabelled rBPI ₂₁Or rBPI (10-193) C132A mixes to the dilution range of 0.01nM with 5 μ M among the DMEM, contain HEPES damping fluid and bovine serum albumin (BSA) [U.S.Biochemicals among this DMEM, Cleveland, OH] and 100 μ L mixtures are joined the Immulon-II plate well (Calbiochem that wraps quilt with 2.5 μ g/mL intestinal bacteria J5LPS in advance, San Diego, CA) in.Flat board was also washed 3 times with the DMEM substratum in 4 ℃ of incubations in 5 hours.Add the NaOH of 75 μ L0.1N and shift out bonded ¹²⁵I-rBPI ₂₁And counting.The result show two kinds of albumen all similarly with radiolabeled rBPI ₂₁Competition.B. form activity in the rate analysis measuring mixture

Compare rBPI (10-193) C132A and rBPl with the speed tuurbidimetry ₂₁LPS in conjunction with active, assessment rBPI ₂₁In conjunction with the method for LPS measure since in the solution light that formation caused of LPS-BPI protein product mixture disperse and advance the speed.All experiments are carried out with BeckmanArray 360 speed turbidimetries (Rate Nephelometer), and the automatic biased sample of this instrument is measured light and dispersed the line speed calculating of going forward side by side.

With the front measuring of this method the dependency measured of best LPS kind and concentration, determination and analysis specificity, repeatability and analytical results and sterilization.Observe intestinal bacteria J5LPS and lipid A and rBPI ₂₁The mixture that formation can be measured in turbidimetry, but intestinal bacteria O111:B4 LPS does not have to form the mixture that can measure.According to these results of study,, select intestinal bacteria J5 LPS with the concentration of 49.4 to 61.7 μ g/ml and the rBPI of 5 to 30 μ g/ml according to the LPS share ₂₁Concentration (in the stream of cells) is united use.The best rBPI that must be measured each LPS share ₂₁Concentration range is about 15 to 25 μ g/ml, and it represents the most linear part of this curve.The optimum range of coalescence rate (RT) value is from 700 to 2000.Comprise PLURONIC P103 or when NaCl concentration increases, need the rBPI of lower concentration when the preparation damping fluid becomes ₂₁To reach identical coalescence rate.Adding is in conjunction with the reorganization lipopolysaccharide binding protein (rLBP of LPS ₅₀) or in conjunction with the effects of heparin rBPI of BPI protein product ₂₁The formation of-LPS aggregation shows this interactional specificity.By BPI that measures many shares and the rBPI that measures same percentage ₂₁The repeatability measured of validating analysis repeatedly.Handle a week and the rBPI of part inactivation by 45 ℃ ₂₁The nephelometric analysis of sample is with consistent dry straightly with the little dilution sterilization of the substratum determination and analysis of intestinal bacteria J5 cell.

Compare rBPI (10-193) C132A and rBPI ₂₁Turbidity measurement experiment be performed as follows.With the LPS[of supersound process intestinal bacteria J5 LPS Lot No.30119B from List Biochemicals] with rBPI (10-193) C132A or rBPI ₂₁[both all use 0.2%PLURONICF68 (poloxamer 188), 0.002%TWEEN 80 (polysorbate fat 80), the Citrate trianion of 5mMpH5.0,150mM NaCl to be prepared] directly dilution goes in the PBS damping fluid of being supplied by Beckman (replenishing with PEG).Fixed L PS concentration but BPI protein product concentration are according to each experiment and change.Measure the LPS:24.7 and the 49.4 μ g/mlLPS of two concentration.Add 42 μ l BPI protein product diluents to the stream of cells and begin each reaction by adding 60 μ l PBS-PEG damping fluids.After setting up baseline, add the intestinal bacteria J5LPS solution of 42 μ l.After adding last a kind of component, measure the speed that mixture forms according to the scope turbidimetry of light diffusion.By with the RT value of each specimen that contains given BPI protein product concentration divided by the corresponding RT value of standard with generation percentage control value and analytical data.For the BPI protein product concentration of each mensuration, measure maximum coalescence rate and set up curve.The data point (equivalent number strong point) that only is positioned at the maximum value left side is used for the different BPI protein product sample of comparative analysis.Standard share by the RT value that relatively is used to measure and curve linear zone and the relative reactivity of working sample.Can use point-to-point or curve appropriate methodology (curve fitapproach).

Except rBPI (10-193) C132A of test purifying and the rBPI of purifying ₂₁[containing about 7.8%rBPI (10-193) C132A] is also to these proteic equal blend things and rBPI with 16%rBPI (10-193) C132A ₂₁Assess (only when 49.4 μ g/ml LPS).When these results are presented at 49.4 μ g/ml LPS, the rBPI of rBPI (10-193) C132A and about 25% lower concentration ₂₁Reach similar coalescence rate.RBPI (10-193) C132A when 27.4 and 49.4 μ g/ml LPS all than rBPI ₂₁Reach higher maximum coalescence rate.The balanced mix deposits yields of two kinds of molecules is between rBPI ₂₁And the curve between the rBPI (10-193) and the rBPI of 7.8% share ₂₁Show consistent each other with the 10-193 of 16% share.This result's point-to-point analysis (49.4 μ g/ml LPS) shows that rBPI (10-193) approximately is rBPI in this analysis ₂₁Active twice.

Embodiment 6

The body outer disinfecting activity of rBPI (10-193) C132A

All assay determinations among this embodiment are all carried out with rBPI (10-193) C132A for preparing in 2 liters of fermentor tanks among the embodiment 3A.A. radiate colibacillary effect in the diffusion analysis

This radiation diffusion analysis is rBPI (10-193) C132A and the rBPI of purifying relatively ₂₁To the bactericidal effect of intestinal bacteria J5, these intestinal bacteria are " rough type " muton of UDP-semi-lactosi-4-epimerase of smooth type coli strain 011B4.With intestinal bacteria J5 cell (Mannion etc., Journal of Clinical Investigation, 85:853-860 (1990); List BiologicalLaboratories, Campbell CA) is cultured to exponential time base, and is centrifugal and with twice of the 10mM sodium phosphate of pH7.4 flushing and with about 1 * 10 ⁶The final concentration of CFU/ml join replenish with 3%Trypticase Soy Broth (TSB, DIFCO Laboratories, Detroit, MI), in the fusion agarose of 10mM sodium phosphate.The aperture of preparation 3mm diameter and in the hardened agarose with the rBPI of 5 μ L serial dilutions ₂₁Or rBPI (10-193) C132A joins in the aperture.Thereby in 37 ℃ of incubations diffusion is taken place flat board, and add the agarose tectum of the fusion that contains 6%TSB then.Flat board is incubated overnight and with the net area that suppresses concentration is mapped in 37 ℃.The result shows (10-193) C132A of rBPI in this analysis and rBPI ₂₁Show similar.B. in the radiation diffusion analysis to the effect of streptococcus aureus L-form

This radiation diffusion analysis is rBPI (10-193) C132A and the rBPI of purifying relatively ₂₁The bactericidal effect of the golden yellow grape grape of the Gram of acellular wall to cultivating into L-shape-positive bacteria coccus.As in U.S. Patent No. 5,578, described in 572 (being incorporated herein for reference), streptococcus aureus L-shape cell is being replenished with 3.5%NaCl, 10mM CaCl ₂Be cultured to logarithmic phase in heart perfusion (HI) substratum of 1000U/mL penicillin G.Cell is diluted to about 5 * 10 in fusion 0.8% agarose that contains the HI substratum that replenishes NaCl ⁴Or 5 * 10 ⁵Individual cell/mL, and 8ml cell-agarose suspension poured in the 10cm flat board.The aperture of preparation 3mm diameter, and with the rBPI of 5 μ L serial dilutions ₂₁Or rBPI (10-193) C132A joins in this aperture.Flat board is mapped to concentration in 37 ℃ of incubations 24 hours and with the net area that suppresses.The result shows rBPI ₂₁And rBPI (10-193) C132A in this is analyzed all in a similar fashion at cell concn about 5 * 10 ⁴With 5 * 10 ⁵The time suppress the growth of golden yellow grape grape coccus L-form.C. in the little dilution analysis of substratum to the effect of intestinal bacteria J5

The little dilution analysis of this substratum is rBPI (10-193) C132A and the rBPI of purifying relatively ₂₁Bactericidal effect to intestinal bacteria J5.Infecting according to former Horwitz etc., immunity, described in the 63:522-527 (1995) with intestinal bacteria J5 cell overnight incubation and in the TEA substratum, be cultured to logarithmic phase then in tryptone yeast extract (TYE) substratum.With cell from about 1 * 10 ⁴With 1 * 10 ⁵Individual cell/mL is inoculated in heart perfusion (HI) substratum, and 95 μ L are joined in the 96 hole flat boards.5 μ L rBPI (10-193) C132A or the rBPI that in the preparation damping fluid, prepare ₂₁Various diluents join in every hole and with flat board in 37 ℃ of incubations 24 hours.The result shows rBPI (10-193) C132A and rBPI ₂₁In analyzing, these have similar activity.

Embodiment 7

Among the external LPS of rBPI (10-193) C132A and active

The analysis of this embodiment A part is used among the embodiment 3A rBPI (10-193) C132A for preparing in 2 liters of fermentor tanks to be carried out, and the analysis in this Embodiment B is carried out with rBPI (10-193) C132A for preparing in 500 liters of fermentor tanks among the embodiment 3B.A. the activity in the RAW analysis of cell proliferation

The RAW analysis of cell proliferation is used for comparison rBPI ₂₁With among the external LPS of rBPI (10-193) C132A and active.In this was analyzed, LPS suppressed the propagation of RAW cell, and rBPI ₂₁In and this effect of LPS.

Mouse RAW 264.7 cells (ATCC license number T1B71) that at first will be maintained in preceding 24 hours in RPMI 1640 substratum (GIBCO) in analysis pass through at 5U/mL recombined small-mouse gamma-interferon (Genzyme, Cambridge, MA) incubation under existing and being induced, RPMI 1640 culture medium supplemented wherein 10mM HEPES damping fluid (pH7.4), the 2mML-glutamine, penicillin (100U/mL), Streptomycin sulphate (100 μ g/mL), 0.075% sodium bicarbonate, 0.15M 2 mercapto ethanol and 10% foetal calf serum (Hyclone, company limited, Logan, UT).Mechanical collection inductive cell and centrifugal and be resuspended in then in 50mL RPMI 1640 substratum (no fill-in) in 4 ℃ of 500 * g then, recentrifuge also is resuspended in RPMI 1640 substratum (no fill-in) once more.Counting cells is adjusted to 2 * 10 with its concentration ⁵Individual cell/mL and adding in each aperture of 100 μ L equal portions to 96 orifice plates.

Then with these cells and intestinal bacteria O113 LPS (Control Standard, Assoc.of Cape Code, Woods Hole, MA) incubation is about 15 hours, and this LPS adds with concentration (this concentration is the result of 50Pg/mL to the titration experiments of 100ng/mL for LPS concentration wherein) the 100 μ L/ hole equal portions of 1ng/mL in serum-free RPMI 1640 substratum.This incubation is at the rBPI of 25ng/mL to 50ng/mL concentration ₂₁Or carry out under rBPI (10-193) C132A existence or the non-existent situation.The people rBPI of reorganization ₂₁, be also referred to as rBPI ₂₁Δ cys (is rBPI _1-93, wherein 132 L-Ala is that halfcystine substitutes [seeing the U.S. Patent No. of owning together 5,420,019]) with the concentration of 1 μ g/mL with comparing.By analyze beginning added in back 5 hours 1 μ Ci/ hole [ ³H]-thymus pyrimidine and quantitative assay cell proliferation.Behind the incubation 15 hours, with cell harvester (cell harvesker) (Inotech Biosystems, INB-384, sample preparation and filter membrane number system, Lansing, MI.) with the cell harvesting of mark to glass fiber filter.The inhibition to RAW 264.7 cell proliferations of LPS-mediation depends on the existence that joins the LBP in the reaction mixture as serum composition or reorganization LBP (with the concentration of 1 μ g/mL).

In these experiments, rBPI ₂₁And rBPI (10-193) C132A all suppresses the inhibition to RAW cell proliferation of LPS-mediation similarly.B. the activity in the TNF inhibition analysis

Tumour necrosis factor (TNF) inhibition analysis is used for comparison rBPI ₂₁And among the external LPS of rBPI (10-193) C132A and active.In this was analyzed, LPS stimulated the TNF of THP-1 cell (human monocyte cell line) to synthesize with the LBP of purifying (or contain LBP serum), and rBPI ₂₁In and this effect of LPS.

(ATCC license number TIB-202) is maintained at the RPMI (GibcoBRL with 10%FBS with the THP.1 cell, Gaithersburg, MD) be incubated at and have 10%FBS and add 50 ng/ml 1 in and handling first three sky with LPS, 25 dihydroxyvitamin Ds (BIOMOLResearch Laboratories company limited, Plymouth Meeting, thereby the expression of inducing CD 14 among RPMI PA).Before inducing with LPS, with cell with RPMI flushing three times and be suspended among the RPMI with 10%FBS or the substratum of serum-free [replenish 1%HB101 RPMI (Irvine Scientific, Santa Ana, CA)] in.In 96 hole flat boards with about 5 * 10 ⁴(Sigma, St.Louis MO) induce the expression of TNF with 1ng/ml intestinal bacteria 0128 LPS in individual cell every hole.With flat board in 37 ℃, 5%CO ₂Following incubation takes out the supernatant liquor five equilibrium and then with CellTiter 96 ^TM(Promega company, Madison is WI) by WEHI 164 oxicity analysis monitoring cell survival for AQ.(GenzymeDiagnostics, Cambridge is MA) as positive criteria for the reorganization human TNF alpha.RBPI ₂₁And rBPI (10-193) C132A the two all suppress the LPS-inductive similarly the TNF synthetic stimulated.

Embodiment 8

Biologic activity in the body of rBPI (10-193) C132A

Be used among the embodiment 3B purifying rBPI (10-193) C132A for preparing in 500 liters of fermentor tanks and carry out following body inner analysis.A. the toxicity research in rat

Compare rBPI in rat ₂₁And the toxicity profile of rBPI (10-193) C132A, in this research, six male and the winding of six female Sprague-Dawley rat experiments is subjected to or vehicle Control (preparation damping fluid), the rBPI of low dosage (50mg/kg/ days) or high dosage (120mg/kg/ days) ₂₁Or rBPI (10-193) C132A.By using these dosage with the continuous intravenously perfusion of the femoral conduit (indwelling femoralcatheter) of the continuous three days implantation of the irrigation rate of 4.2mL/lkg/ hour (100mL/kg/ days).Clinical observation result writes down twice and writes down every day body weight every day at least.Collect the blood urine sample during near end and be used for hematology, clinical chemistry and urinalysis mensuration.During end, weighing organ and collection organization are used for histopathological examination.Do not have death or significantly test article (test article) related effect.These data show rBPI when imposing continous pouring ₂₁Has similar toxicity profile with rBPI (10-193) C132A.B. pharmacokinetics

The rBPI of 2mg/kg ₂₁Study in rat with the pharmacokinetics of rBPI (10-193) C132A.RBPI ₂₁Handle function (tri-exponential pharmacokinetic dispositionfunction) with the plasma clearance of rBPI (10-193) C132A by three-index pharmacokinetics and obtain fine description.Do not detect the statistical difference opposite sex (distribution free Wicoxon rank check, p＜0.05) in the rBPI product pharmacokinetic parameter.The medicine that great majority are used (＞96%) obtained removing in the β half life of 0.2-0.4 minute half life α phase and 3.9-4.3 minute, and rest part obtained removing during the γ phase of 27-33 minute half life.The volume (Vc) that middle body (central compartment) distributes is 41-45mg/kg, and clearance rate (CL) is 24-30mL/ minute/kg.The stable state volume of this distribution is 152-184mL/kg.C. the efficient in mouse intracellular toxin inoculation

Basically according to Ammons etc., at " the new therapeutic strategy of treatment sepsis ", Morrison and Ryan compile, Marcel Dekker, New York (1996), the 55-69 page or leaf mouse cause death endotoxemia (endotoxemia) thus carry out twice independent research in the model and measure rBPI ₂₁Relative vigor with rBPI (10-193) C132A.In two researchs, in each processing and control group, 14 mouse are arranged.In first research, the intestinal bacteria O111 of inoculation 25mg/kg in the CD1 mouse vein: the lipopolysaccharides of B4 (LPS).After the inoculation immediately with mouse with 15,20,25 and the rBPI of the dosage of 30mg/kg ₂₁Or rBPI (10-193) C132A or the processing of control vector (only preparing damping fluid) intravenously.Continuous seven day every day twice record lethality rate.

First result of study that is shown in the following table 1 shows with rBPI (10-193) C132A and rBPI ₂₁Handle and all significantly increased survival than vehicle Control.In addition, rBPI (10-193) C132A is than rBPI ₂₁At least the high twice of efficient is because than rBPI ₂₁See dosage rBPI therewith among rBPI (10-193) C132A of low two multiple doses ₂₁Similar survival efficient.

Table 1

	15 numbers of merely hitting and surviving
				Dosage (mg/kg)	Contrast	????rBPI 21	????rBPI(10- ????193)C132A
0 (carrier)	????0	????NA 1	????NA
				????15		????0	????15 **.##
????20		????4	????15 **,#
				????25		????11 **	????15 **
????30		????13 **	????13 **

¹NA, inapplicable ^*, with contrast ratio p＜0.01#, with rBPI ₂₁Than p＜0.05##, with contrast ratio p＜0.01

In second research, studied more the dosage of the rBPI of wide region (10-193) C132A (5,10,15,20,25,30mg/kg).Although it is the same with first research that the result shown in the following table confirms, rBPI ₂₁All obtained tangible survival effect with rBPI (10-193) C132A than contrast, but rBPI (10-193) the C132A high twice of efficient at least, because obtain and rBPI with the dosage that is lower than 2 times ₂₁Similar efficient.

Table 2

	15 numbers of merely hitting and surviving
				Dosage (mg/kg)	Contrast	????rBPI 21	????rBPI(10- ????193)C132A
0 (carrier)	????2	????NA 1	????NA
				????5		????ND 1	????3
????10		????ND	????10 *
				????15		????ND	????14 **
????20		????7	????14 **,#
				????25		????13 **	????15 **
????30		????14 **	????15 **

¹NA, inapplicable; ND does not carry out ^*, with contrast ratio p＜0.05 ^*, with contrast ratio p＜0.01#, with rBPI ₂₁Than the efficient of p＜0.05D. in the microbemia mouse model that causes death

Thereby carried out two independent researchs and measured rBPI ₂₁With the relative efficiency of rBPI (10-193) C132A in the microbemia mouse model that causes death.In first research, CD 1 mouse is used by intravenously and inoculates with 6.8 * 10 ⁷Individual intestinal bacteria 07: the colony-forming unit of K1 (CFU).Use 10,20 and the rBPI of 30mg/kg dosage after the inoculation immediately ₂₁Or rBPI (10-193) C132A or control vector (only preparing damping fluid) are handled mouse by intravenously.Continuous seven day every day twice record lethality rate.

Being shown in first result of study in the following table 3 shows with 10 and 30mg/kg rBPI ₂₁The significantly increase of handling of group survival (p＜0.05, compared with the control).Significantly do not increase rBPI in this research although rBPI (10-193) C132A observes survival compared with the control ₂₁And the survival advantage does not have significant difference between the group that rBPI (10-193) C132A handles.

Table 3

	20 numbers of merely hitting and surviving
				Dosage (mg/kg)	Contrast	????rBPI 21	????rBPI(10- ????193)C132A
0 (carrier)	????6		????NA
				????10		????14 *	????12
????20		????12	????10
				????30		????14 *	????10

^*, p＜0.05 compared with the control

For more fully to rBPI ₂₁Carry out signature analysis with the effect of rBPI (10-193) C132A in this model, carry out second experiment with dosage range more widely.In this research, CD 1 mouse is used by intravenously and inoculates 2.57 * 10 ⁸Individual intestinal bacteria 07: K1 colony-forming unit (CFU).Use 1.0,3.0,10 and 30mg/kg rBPI after the inoculation immediately ₂₁And 0.3,1.0,3.0,10 and 30mg/kg rBPI (10-193) C132A intravenously handle mouse.The result who is shown in the following table 4 shows that both all provide protection, and the protective effect no significant difference between two kinds of varients of any dosage.

Table 4

	20 numbers of merely hitting and surviving
				Dosage (mg/kg)	Contrast	????rBPI 21	????rBPI(10- ????193)C132A
0 (carrier)	????6
				????0.3		????ND 1	????6
????1.0		????4	????6
				????3.0		????9	????10 *
????10		????13 **	????8
				????30		????11 *	????14 **

¹ND does not measure ^*, p＜0.05 compared with the control ^*, the cardiovascular effect of p＜0.01E. in rat consciously compared with the control

Thereby carry out a series of experiments and measure rBPI ₂₁With the relative effect of rBPI (10-193) C132A to rat blood pressure.(Fort Dodge Labs, Fort Dodge is IA) with Rompum (Bayer company, Shawnee Mission, every rat of mixture anesthesia KS) with Ketamine.Then a conduit is placed right carotid artery and with blood pressure conduction only (pressuretransducer) be connected with recording blood pressure.Place right jugular vein with injection rBPI or carrier in second conduit.Before the experiment beginning, allow rat recover then.When become vigilance, movable and blood pressure stabilization of rat begins experiment in normal range the time.Then with rBPI ₂₁, rBPI (10-193) C132A or control vector (preparation damping fluid) 60 minutes mean arterial pressure (mmHg) as bolus and after injection in 15 seconds and record.

In preliminary experiment, record the rBPI of 20 and 30 mg/kg dosage ₂₁Than carrier blood pressure is not had tangible effect, but the dosage of 40mg/kg obtained the significant decline of blood pressure in 5 minutes.This hypotension reached maximum in back 15 minutes in injection, and this moment, blood pressure reduced by 48 ± 12mmHg (mean value ± SE; P＞0.05).After 60 minutes, rBPI ₂₁-the blood pressure of handling animal recovers and does not have significant difference with the blood pressure of vehicle treated animal.

In order to compare rBPI ₂₁With the effect of rBPI (10-193) C132A, the experimental group of 5 rats is given every kind of drug substance or the vehicle Control of 40mg/kg, and is area under curve (AUC) with the blood pressure response analysis.With in the past observed the same, Fig. 1 shows rBPI ₂₁Cause blood pressure to descend significantly, show as the AUC that raises than control vector.By relatively, compare with vehicle Control, rBPI (10-193) C132A does not have obvious influence to blood pressure.The rBPI of 50mg/kg dosage ₂₁(N=4 rat) has bigger ypotension effect than the dosage of 40mg/kg, shows as the further rising of AUC among Fig. 1.When this higher dosage, though the decline of some blood pressures also occurs in the rat (N=3) of using rBPI (10-193) C132A, this effect is compared not obvious with vehicle Control.

The invention described above of numerous modifications and variation to to(for) those skilled in the art are estimated and can be taken place.Therefore, only those just should be placed in one as the qualification that appears in the appended claims.

Sequence table＜110〉XOMA technology company

Horwitz, Arnold (contriver)

Carroll, Stephen F. (contriver)

Burke; David (inventor)＜120〉sterilization/saturating property-increase albumen (BPI) delation analogs＜130〉29715/35765＜140〉＜141＜150〉09/099,725＜151〉1998-06-19＜160〉6＜170〉PatentIn Ver.2.0＜210〉1＜211〉1813＜212〉DNA＜213〉people＜220〉＜221〉CDS＜222〉(31) .. (1491)＜220〉＜221〉mature peptide＜222〉(124) .. (1491)＜220〉＜223〉rBPI＜400〉1caggccttga ggttttggca gctctggagg atg aga gag aac atg gcc agg ggc 54

Met?Arg?Glu?Asn?Met?Ala?Arg?Gly

-30?????????????????-25cct?tgc?aac?gcg?ccg?aga?tgg?gtg?tcc?ctg?atg?gtg?ctc?gtc?gcc?ata???102Pro?Cys?Asn?Ala?Pro?Arg?Trp?Val?Ser?Leu?Met?Val?Leu?Val?Ala?Ile

-20?????????????????-15?????????????????-10ggc?acc?gcc?gtg?aca?gcg?gcc?gtc?aac?cct?ggc?gtc?gtg?gtc?agg?atc???150Gly?Thr?Ala?Val?Thr?Ala?Ala?Val?Asn?Pro?Gly?Val?Val?Val?Arg?Ile

-5??????????????-1???1???????????????5tcc?cag?aag?ggc?ctg?gac?tac?gcc?agc?cag?cag?ggg?acg?gcc?gct?ctg???198Ser?Gln?Lys?Gly?Leu?Asp?Tyr?Ala?Ser?Gln?Gln?Gly?Thr?Ala?Ala?Leu?10??????????????????15??????????????????20??????????????????25cag?aag?gag?ctg?aag?agg?atc?aag?att?cct?gac?tac?tca?gac?agc?ttt???246Gln?Lys?Glu?Leu?Lys?Arg?Ile?Lys?Ile?Pro?Asp?Tyr?Ser?Asp?Ser?Phe

30??????????????????35??????????????????40aag?atc?aag?cat?ctt?ggg?aag?ggg?cat?tat?agc?ttc?tac?agc?atg?gac???294Lys?Ile?Lys?His?Leu?Gly?Lys?Gly?His?Tyr?Ser?Phe?Tyr?Ser?Met?Asp

45??????????????????50??????????????????55atc?cgt?gaa?ttc?cag?ctt?ccc?agt?tcc?cag?ata?agc?atg?gtg?ccc?aat???342Ile?Arg?Glu?Phe?Gln?Leu?Pro?Ser?Ser?Gln?Ile?Set?Met?Val?Pro?Asn

60??????????????????65??????????????????70gtg?ggc?ctt?aag?ttc?tcc?atc?agc?aac?gcc?aat?atc?aag?atc?agc?ggg???390Val?Gly?Leu?Lys?Phe?Ser?Ile?Ser?Asn?Ala?Asn?Ile?Lys?Ile?Ser?Gly

75??????????????????80??????????????????????????85aaa?tgg?aag?gca?caa?aag?aga?ttc?tta?aaa?atg?agc?ggc?aat?ttt?gac???438Lys?Trp?Lys?Ala?Gln?Lys?Arg?Phe?Leu?Lys?Met?Ser?Gly?Asn?Phe?Asp?90??????????????????95?????????????????100?????????????????105ctg?agc?ata?gaa?ggc?atg?tcc?att?tcg?gct?gat?ctg?aag?ctg?ggc?agt???486Leu?Ser?Ile?Glu?Gly?Met?Ser?Ile?Ser?Ala?Asp?Leu?Lys?Leu?Gly?Ser

110?????????????????115?????????????????120aac?ccc?acg?tca?ggc?aag?ccc?acc?atc?acc?tgc?tcc?agc?tgc?agc?agc???534Asn?Pro?Thr?Ser?Gly?Lys?Pro?Thr?Ile?Thr?Cys?Ser?Ser?Cys?Ser?Ser

125?????????????????130?????????????????135cac?atc?aac?agt?gtc?cac?gtg?cac?atc?tca?aag?agc?aaa?gtc?ggg?tgg???582His?Ile?Asn?Ser?Val?His?Val?His?Ile?Ser?Lys?Ser?Lys?Val?Gly?Trp

140?????????????????145?????????????????150ctg?atc?caa?ctc?ttc?cac?aaa?aaa?att?gag?tct?gcg?ctt?cga?aac?aag???630Leu?Ile?Gln?Leu?Phe?His?Lys?Lys?Ile?Glu?Ser?Ala?Leu?Arg?Asn?Lys

155?????????????????160?????????????????165atg?aac?agc?cag?gtc?tgc?gag?aaa?gtg?acc?aat?tct?gta?tcc?tcc?aag???678Met?Asn?Ser?Gln?Val?Cys?Glu?Lys?Val?Thr?Asn?Ser?Val?Ser?Ser?Lys170?????????????????175?????????????????180?????????????????185ctg?caa?cct?tat?ttc?cag?act?ctg?cca?gta?atg?acc?aaa?ata?gat?tct???726Leu?Gln?Pro?Tyr?Phe?Gln?Thr?Leu?Pro?Val?Met?Thr?Lys?Ile?Asp?Ser

190?????????????????195?????????????????200gtg?gct?gga?atc?aac?tat?ggt?ctg?gtg?gca?cct?cca?gca?acc?acg?gct???774Val?Ala?Gly?Ile?Asn?Tyr?Gly?Leu?Val?Ala?Pro?Pro?Ala?Thr?Thr?Ala

205?????????????????210?????????????????215gag?acc?ctg?gat?gta?cag?atg?aag?ggg?gag?ttt?tac?agt?gag?aac?cac???822Glu?Thr?Leu?Asp?Val?Gln?Met?Lys?Gly?Glu?Phe?Tyr?Ser?Glu?Asn?His

220?????????????????225?????????????????230cac?aat?cca?cct?ccc?ttt?gct?cca?cca?gtg?atg?gag?ttt?ccc?gct?gcc???870His?Asn?Pro?Pro?Pro?Phe?Ala?Pro?Pro?Val?Met?Glu?Phe?Pro?Ala?Ala

235?????????????????240?????????????????245cat?gac?cgc?atg?gta?tac?ctg?ggc?ctc?tca?gac?tac?ttc?ttc?aac?aca???918His?Asp?Arg?Met?Val?Tyr?Leu?Gly?Leu?Ser?Asp?Tyr?Phe?Phe?Ash?Thr250?????????????????255?????????????????260?????????????????265gcc?ggg?ctt?gta?tac?caa?gag?gct?ggg?gtc?ttg?aag?atg?acc?ctt?aga???966Ala?Gly?Leu?Val?Tyr?Gln?Glu?Ala?Gly?Val?Leu?Lys?Met?Thr?Leu?Arg

270?????????????????275?????????????????280gat?gac?atg?att?cca?aag?gag?tcc?aaa?ttt?cga?ctg?aca?acc?aag?ttc???1014Asp?Asp?Met?Ile?Pro?Lys?Glu?Ser?Lys?Phe?Arg?Leu?Thr?Thr?Lys?Phe

285?????????????????290?????????????????295ttt?gga?acc?ttc?cta?cct?gag?gtg?gcc?aag?aag?ttt?ccc?aac?atg?aag???1062Phe?Gly?Thr?Phe?Leu?Pro?Glu?Val?Ala?Lys?Lys?Phe?Pro?Ash?Met?Lys

300?????????????????305?????????????????310ata?cag?atc?cat?gtc?tca?gcc?tcc?acc?ccg?cca?cac?ctg?tct?gtg?cag???1110Ile?Gln?Ile?His?Val?Ser?Ala?Ser?Thr?Pro?Pro?His?Leu?Ser?Val?Gln

315?????????????????320?????????????????325ccc?acc?ggc?ctt?acc?ttc?tac?cct?gcc?gtg?gat?gtc?cag?gcc?ttt?gcc???1158Pro?Thr?Gly?Leu?Thr?Phe?Tyr?Pro?Ala?Val?Asp?Val?Gln?Ala?Phe?Ala330?????????????????335?????????????????340?????????????????345gtc?ctc?ccc?aac?tcc?tcc?ctg?gct?tcc?ctc?ttc?ctg?att?ggc?atg?cac???1206Val?Leu?Pro?Asn?Ser?Ser?Leu?Ala?Ser?Leu?Phs?Leu?Ile?Gly?Met?His

350?????????????355?????????????????360aca?act?ggt?tcc?atg?gag?gtc?agc?gcc?gag?tcc?aac?agg?ctt?gtt?gga???1254Thr?Thr?Gly?Ser?Met?Glu?Val?Ser?Ala?Glu?Ser?Asn?Arg?Leu?Val?Gly

365?????????????????370?????????????????375gag?ctc?aag?ctg?gat?agg?ctg?ctc?ctg?gaa?ctg?aag?cac?tca?aat?att???1302Glu?Leu?Lys?Leu?Asp?Arg?Leu?Leu?Leu?Glu?Leu?Lys?His?Ser?Asn?Ile

380?????????????????????385?????????????390ggc?ccc?ttc?ccg?gtt?gaa?ttg?ctg?cag?gat?atc?atg?aac?tac?att?gta???1350Gly?Pro?Phe?Pro?Val?Glu?Leu?Leu?Gln?Asp?Ile?Met?Ash?Tyr?Ile?Val

395?????????????????????400?????????????405ccc?att?ctt?gtg?ctg?ccc?agg?gtt?aac?gag?aaa?cta?cag?aaa?ggc?ttc???1398Pro?Ile?Leu?Val?Leu?Pro?Arg?Val?Asn?Glu?Lys?Leu?Gln?Lys?Gly?Phe410?????????????????415?????????????????420?????????????????425cct?ctc?ccg?acg?ccg?gcc?aga?gtc?cag?ctc?tac?aac?gta?gtg?ctt?cag???1446Pro?Leu?Pro?Thr?Pro?Ala?Arg?Val?Gln?Leu?Tyr?Asn?Val?Val?Leu?Gln

430?????????????????435?????????????????440cct?Gac?Gag?aac?ttc?ctg?ctg?ttc?ggt?gca?gac?gtt?gtc?tat?aaa????????1491Pro?His?Gln?Asn?Phe?Leu?Leu?Phe?Gly?Ala?Asp?Val?Val?Tyr?Lys

445 450 455tgaaggcacc aggggtgccg ggggctgtca gccgcacctg ttcctgatgg gctgtggggc 1551accggctgcc tttccccagg gaatcctctc cagatcttaa ccaagagccc cttgcaaact 1611tcttcgactc agattcagaa atgatctaaa cacgaggaaa cattattcat tggaaaagtg 1671catggtgtgt attttaggga ttatgagctt ctttcaaggg ctaaggctgc agagatattt 1731cctccaggaa tcgtgtttca attgtaacca agaaatttcc atttgtgctt catgaaaaaa 1791aacttctggt ttttttcatg tg, 1813＜210〉2＜211〉487＜212〉protein＜213〉people＜400〉2 Met Arg Glu Asn Met Ala Arg Gly Pro Cys Asn Ala Pro Arg Trp Val

-30?????????????????-25?????????????????-20?Ser?Leu?Met?Val?Leu?Val?Ala?Ile?Gly?Thr?Ala?Val?Thr?Ala?Ala?Val?-15?????????????????-10??????????????????-5??????????????-1???1?Asn?Pro?Gly?Val?Val?Val?Arg?Ile-Ser?Gln?Lys?Gly?Leu?Asp?Tyr?Ala

5??????????????????10??????????????????15?Ser?Gln?Gln?Gly?Thr?Ala?Ala?Leu?Gln?Lys?Glu?Leu?Lys?Arg?Ile?Lys

20??????????????????25??????????????????30?Ile?Pro?Asp?Tyr?Ser?Asp?Ser?Phe?Lys?Ile?Lys?His?Leu?Gly?Lys?Gly

35??????????????????40??????????????????45?His?Tyr?Ser?Phe?Tyr?Ser?Met?Asp?Ile?Arg?Glu?Phe?Gln?Leu?Pro?Ser???50??????????????????55??????????????????60??????????????????65?Ser?Gln?Ile?Ser?Met?Val?Pro?Asn?Val?Gly?Leu?Lys?Phe?Ser?Ile?Ser

70??????????????????75??????????????????80?Asn?Ala?Asn?Ile?Lys?Ile?Ser?Gly?Lys?Trp?Lys?Ala?Gln?Lys?Arg?Phe

80??????????????????90??????????????????95?Leu?Lys?Met?Ser?Gly?Asn?Phe?Asp?Leu?Ser?Ile?Glu?Gly?Met?Ser?Ile

100?????????????????105?????????????????110?Ser?Ala?Asp?Leu?Lys?Leu?Gly?Ser?Asn?Pro?Thr?Ser?Gly?Lys?Pro?Thr

115?????????????????120?????????????????125?Ile?Thr?Cys?Ser?Ser?Cys?Ser?Ser?His?Ile?Asn?Ser?Val?His?Val?His?130?????????????????135?????????????????140?????????????????145?Ile?Ser?Lys?Ser?Lys?Val?Gly?Trp?Leu?Ile?Gln?Leu?Phe?His?Lys?Lys

150?????????????????155?????????????????160?Ile?Glu?Ser?Ala?Leu?Arg?Asn?Lys?Met?Asn?Ser?Gln?Val?Cys?Glu?Lys

165?????????????????170?????????????????175Val?Thr?Asn?Ser?Val?Ser?Ser?Lys?Leu?Gln?Pro?Tyr?Phe?Gln?Thr?Leu

180?????????????????185?????????????????190Pro?Val?Met?Thr?Lys?Ile?Asp?Ser?Val?Ala?Gly?Ile?Asn?Tyr?Gly?Leu

195?????????????????200?????????????????205Val?Ala?Pro?Pro?Ala?Thr?Thr?Ala?Glu?Thr?Leu?Asp?Val?Gln?Met?Lys210?????????????????215?????????????????220?????????????????225Gly?Glu?Phe?Tyr?Ser?Glu?Asn?His?His?Asn?Pro?Pro?Pro?Phe?Ala?Pro

230?????????????????235?????????????????240Pro?Val?Met?Glu?Phe?Pro?Ala?Ala?His?Asp?Arg?Met?Val?Tyr?Leu?Gly

245?????????????????250?????????????????255Leu?Ser?Asp?Tyr?Phe?Phe?Asn?Thr?Ala?Gly?Leu?Val?Tyr?Gln?Glu?Ala

260?????????????????265?????????????????270Gly?Val?Leu?Lys?Mer?Thr?Leu?Arg?Asp?Asp?Met?Ile?Pro?Lys?Glu?Ser

275?????????????????280?????????????????285Lys?Phe?Arg?Leu?Thr?Thr?Lys?Phe?Phe?Gly?Thr?Phe?Leu?Pro?Glu?Val290?????????????????295?????????????????300?????????????????305Ala?Lys?Lys?Phe?Pro?Asn?Met?Lye?Ile?Gln?Ile?His?Val?Ser?Ala?Ser

310?????????????????315?????????????????320Thr?Pro?Pro?His?Leu?Ser?Val?Gln?Pro?Thr?Gly?Leu?Thr?Phe?Tyr?Pro

325?????????????????330?????????????????335Ala?Val?Asp?Val?Gln?Ala?Phe?Ala?Val?Leu?Pro?Asn?Ser?Ser?Leu?Ala

340?????????????????345?????????????????350Ser?Leu?Phe?Leu?Ile?Gly?Met?His?Thr?Thr?Gly?Ser?Met?Glu?Val?Ser

355?????????????????360?????????????????365Ala?Glu?Ser?Asn?Arg?Leu?Val?Gly?Glu?Leu?Lys?Leu?Asp?Arg?Leu?Leu370?????????????????375?????????????????380?????????????????385Leu?Glu?Leu?Lys?His?Ser?Ash?Ile?Gly?Pro?Phe?Pro?Val?Glu?Leu?Leu

390?????????????????395?????????????????400Gln?Asp?Ile?Met?Asn?Tyr?Ile?Val?Pro?Ile?Leu?Val?Leu?Pro?Arg?Val

405?????????????????410?????????????????415Asn?Glu?Lys?Leu?Gln?Lys?Gly?Phe?Pro?Leu?Pro?Thr?Pro?Ala?Arg?Val

420?????????????????425?????????????????430Gln?Leu?Tyr?Asn?Val?Val?Leu?Gln?Pro?His?Gln?Asn?Phe?Leu?Leu?Phe

435 440 445Gly Ala Asp Val Val Tyr Lys450 455＜210〉3＜211〉21＜212〉DNA＜213〉artificial sequence＜220〉＜223〉artificial sequence description: primer＜400〉3ctgctctaaa agctgctgca g 21＜210〉4＜211〉33＜212〉DNA＜213〉artificial sequence＜220〉＜223〉artificial sequence description: primer＜400〉4ccaggccctt ctgggaggcc gctgtcacgg cgg 33＜210〉5＜211〉33＜212〉DNA＜213〉artificial sequence＜220〉＜223〉artificial sequence description: primer＜400〉5gccgtgacag cggcctccca gaagggcctg gac 33＜210〉6＜211〉18＜212〉DNA＜213〉artificial sequence＜220〉＜223〉artificial sequence description: primer＜400〉6ctgggaactg ggaagctg 18

Claims (19)

1. one kind by becoming acquaintance BPI amino-acid residue 10 to 193 sterilization/permeability-increasing protein of being formed (BPI) delation analogs, and wherein half deamination of position 132 acid residue is substituted by different amino acid.

2. the BPI delation analogs of claim 1, the amino acid that wherein replaces described cysteine residues is the nonpolar amino acid that is selected from L-Ala or Serine.

3. the BPI delation analogs of claim 1, the cysteine residues of position 132 is wherein substituted by L-Ala.

4. the polynucleotide of the BPI delation analogs of the claim 1 of encoding.

5. the polynucleotide of the BPI delation analogs of the claim 3 of encoding.

6. the polynucleotide of claim 4 further comprise 27 amino acid whose leader sequences of BPI.

7. the polynucleotide of claim 4, it is DNA.

8. the expression vector that comprises claim 7DNA.

9. host cell, it is with DNA stable conversion or the transfection with claim 7 of the mode that allows described polynucleotide delation analogs and express in described host cell.

10. the eukaryotic host cell of claim 9.

11. the host cell of claim 10, it is a Chinese hamster ovary celI.

12. prepare the method for BPI delation analogs polypeptide, be included in the suitable substratum host cell of cultivating claim 9 and from described host cell or described substratum, separate described polypeptide.

13. the polypeptide product of claim 12 method.

14. can accept the composition of thinner, adjuvant or carrier on BPI delation analogs that comprises claim 1 and the medicine.

15. one kind comprises the BPI delation analogs of claim 3 and the composition of pharmaceutically acceptable thinner, adjuvant or carrier.

16. one kind comprises the BPI delation analogs of claim 13 and the composition of pharmaceutically acceptable thinner, adjuvant or carrier.

17. an improved method of using the BPI protein product to the experimenter comprises that the composition of using claim 14 is to described experimenter.

18. an improved method of using the BPI protein product to the experimenter comprises that the composition of using claim 15 is to described experimenter.

19. an improved method of using the BPI protein product to the experimenter comprises that the composition of using claim 16 is to described experimenter.

Images